Methods and compositions for rapid assembly of genetic modules

ABSTRACT

Provided herein are methods and compositions for rapid assembly of genetic modules, as well as seamless transition from in vitro to in vivo testing of genetic constructs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 62/153,308 filed Apr. 27, 2015, the disclosure of whichis hereby incorporated by reference in its entirety.

STATEMENT REGARDING GOVERNMENT LICENSE RIGHTS

This invention was made with government support under contract numberHR0011-12-C-0065 awarded by the U.S. Defense Advanced Research ProjectsAgency (DARPA) Living Foundries Program. The government has certainrights in the invention.

FIELD

The disclosure relates to methods and compositions for rapid in vitroassembly of genetic modules, in particular pre-made DNA modules. Theassembly technique disclosed herein enables seamless transition from invitro to in vivo testing of genetic constructs.

BACKGROUND

Synthetic biology has emerged as a useful approach to decodingfundamental laws underlying biological control. Recent efforts haveproduced many systems and approaches and generated substantial insightson how to engineer biological functions and efficiently optimizesynthetic pathways.

Despite efforts and progresses, current approaches to perform suchengineering are often laborious, costly and difficult. Challenges stillremain in developing engineering-driven approaches and systems toaccelerate the design-build-test cycles required for reprogrammingexisting biological systems, constructing new biological systems andtesting genetic circuits for transformative future applications indiverse areas including biology, engineering, green chemistry,agriculture and medicine.

An in vitro transcription-translation (TX-TL) system (Shin & Noireaux,2012; Sun et al., 2013) has been developed which allows for the rapidprototyping of genetic constructs (Sun, et al., 2014) in an environmentthat behaves similarly to a cell (Niederholtmeyer et al., 2015;Takahashi et al., 2015). One of the main purposes of working in vitro isto be able to learn or characterize a circuit for future implementationin vivo (Chappell et al., 2013; Niederholtmeyer et al., 2015). However,there are no easy ways to convert deoxyribonucleic acid (DNA), which wascreated primarily for in vitro testing, to make the DNA compatible forthe in vivo environment when implemented on plasmid. In specific,origins of replication need to be in compatible families, and antibioticresistance markers need to be varied per plasmid. Thus, a need existsfor new techniques that can overcome these challenges.

SUMMARY

Provided herein are methods and compositions for rapid assembly ofgenetic modules, as well as seamless transition from in vitro to in vivotesting of genetic constructs.

In one aspect, a method for in vitro assembly of genetic modules isprovided, comprising:

-   -   a) providing recombinant transcription units Tu1, Tu2 and TuN        wherein N>=3, each recombinant transcription unit being present        in a separate stage 1 vector and flanked by a first pair of        restriction sites of a first type IIs enzyme, wherein the first        pair of restriction sites for each recombinant transcription        unit are pre-designed such that upon digestion by the first type        IIs enzyme, compatible cohesive ends are generated to allow        ligation of the recombinant transcription units in a        predetermined order 5′-Tu1-TuN-Tu2-3′;    -   b) providing a stage 2 vector having a second pair of        restriction sites of the first type IIs enzyme, wherein the        second pair of restriction sites are pre-designed such that upon        digestion by the first type IIs enzyme, a first and second        cohesive end are generated to allow ligation of the first        cohesive end with Tu1 at its 5′ end and ligation of the second        cohesive end with Tu2 at its 3′ end; and    -   c) assembling the recombinant transcription units and the stage        2 vector into a plasmid in a one-pot reaction comprising the        first type IIs enzyme and a ligase.

Another aspect relates to a method for in vitro assembly of geneticmodules, comprising:

-   -   a) providing recombinant transcription units Tu1 and Tu2, each        recombinant transcription unit being present in a separate stage        1 vector and flanked by a first pair of restriction sites of a        first type IIs enzyme, wherein the first pair of restriction        sites for each recombinant transcription unit are pre-designed        such that upon digestion by the first type IIs enzyme,        compatible cohesive ends are generated to allow ligation of the        recombinant transcription units in a predetermined order        5′-Tu1-Tu2-3′;    -   b) providing a stage 2 vector having a second pair of        restriction sites of the first type IIs enzyme, wherein the        second pair of restriction sites are pre-designed such that upon        digestion by the first type IIs enzyme, a first and second        cohesive end are generated to allow ligation of the first        cohesive end with Tu1 at its 5′ end and ligation of the second        cohesive end with Tu2 at its 3′ end;    -   c) assembling the recombinant transcription units and the stage        2 vector into a plasmid in a one-pot reaction comprising the        first type IIs enzyme and a ligase; and    -   d) amplifying the recombinant transcription units in a        polymerase chain reaction, using a first and second primer that        span the first and second cohesive end, respectively, wherein        the first primer partially anneals with the stage 2 vector at        Tm<40° C. and partially with Tu1 at Tm<40° C., and the second        primer partially anneals with the stage 2 vector at Tm<40° C.        and partially with Tu2 at Tm<40° C.

A further aspect relates to a method for in vitro assembly of geneticmodules, comprising:

-   -   a) providing recombinant transcription units Tu1, Tu2 and TuN        wherein N>=3, each recombinant transcription unit being present        in a separate stage 1 vector and flanked by a first pair of        restriction sites of a first type IIs enzyme, wherein the first        pair of restriction sites for each recombinant transcription        unit are pre-designed such that upon digestion by the first type        IIs enzyme, compatible cohesive ends are generated to allow        ligation of the recombinant transcription units in a        predetermined order 5′-Tu1-TuN-Tu2-3′;    -   b) providing a stage 2 vector having a second pair of        restriction sites of the first type IIs enzyme, wherein the        second pair of restriction sites are pre-designed such that upon        digestion by the first type IIs enzyme, a first and second        cohesive end are generated to allow ligation of the first        cohesive end with Tu1 at its 5′ end and ligation of the second        cohesive end with Tu2 at its 3′ end;    -   c) assembling the recombinant transcription units and the stage        2 vector into a plasmid in a one-pot reaction comprising the        first type IIs enzyme and a ligase; and    -   d) subjecting the plasmid to expression selected from:        -   in vitro expression in an in vitro transcription-translation            system, and/or        -   in vivo expression following transformation into a host            cell.

In some embodiments in connection with any methods for in vitro assemblydisclosed herein, N can be an integer between, and inclusive of, 3 and9, i.e., 3, 4, 5, 6, 7, 8, or 9. For example, N can be 3, 4, 5 or 6.

In various embodiments, the first type IIs enzyme may be selected fromBasI, Eco31I, BspTN1, Bso31I, BbsI, BpuAI, BpiI, BstV21, BsmBI, Esp3I,FokI, AlwI, and BfilI. In some embodiments, the directionality of thefirst pair of restriction sites can be in tandem or opposing (convergingor diverging). In one example, the first pair of restriction sites canbe designed to oppose each other in a converging direction.

The method can, in some embodiments, further include constructing eachrecombinant transcription unit from a promoter, an untranslated region,a coding sequence and a terminator, wherein one or more of the promoter,untranslated region, coding sequence and terminator are provided from alibrary of modular components. Each modular component can be designedand engineered to have flanking restriction sites of a second type IIsenzyme. The second type IIs enzyme can be selected from BsaI, Eco31I,BspTN1, Bso31I, BbsI, BpuAI, BpiI, BstV21, BsmBI, Esp3I, FokI, AlwI, andBfilI. The method may further include selecting the one or more of thepromoter, untranslated region, coding sequence and terminator from thelibrary.

In certain embodiments, each stage 1 vector may include a differentorigin of replication and/or a different selectable marker. Two or moreof the stage 1 vectors may have the same origin of replication and/orthe same selectable marker. Any origins of replication commonly used inmolecular cloning may be used, such as colE1, pSC101, p15A, pBBR1, pMB1and R6K. The selectable marker can be any marker commonly used inmolecular cloning, such as AmpR, KanR, CmR, ZeoR, TetR, SpecR, StrepR,NeoR, and BleR.

The directionality of the second pair of restriction sites in the stage2 vector can be in tandem or opposing (converging or diverging). In oneexample, the second pair of restriction sites can be designed to opposeeach other in a diverging direction. In some embodiments, the stage 2vector can include an additional pair of restriction sites of a secondtype IIs enzyme that flank the second pair of restriction sites of thefirst type IIs enzyme. The second type IIs enzyme can be selected fromBsaI, Eco31I, BspTN1, Bso31I, BbsI, BpuAI, BpiI, BstV21, BsmBI, Esp3I,FokI, AlwI, and BfilI.

The method can further include a step of cycling the recombinanttranscription units between the stage 1 and stage 2 vectors to produce 2or more copies of the recombinant transcription units.

The method can, in some embodiments, further include providing two ormore stage 2 vectors in step (b), and assembling in step (c) therecombinant transcription units and the two or more stage 2 vectors intotwo or more plasmids. Each stage 2 vector may have the same or differentorigin of replication and/or selectable marker. In some embodiments, upto three plasmids can be assembled in step (c) for, e.g.,transformation.

In various embodiments, the plasmid assembled can be subjected toexpression. The plasmid may be subjected to expression in an in vitrotranscription-translation system. The plasmid may also be subjected toin vivo expression following transformation into a host cell. Tofacilitate in vitro expression, it may be desirable to amplify therecombinant transcription units in a polymerase chain reaction (PCR)prior to expression. In some embodiments, specific PCR primers can bedesigned, such as a first and second primer that span the first andsecond cohesive end, respectively, wherein the first primer partiallyanneals with the stage 2 vector at Tm<40° C. and partially with Tu1 atTm<40° C., and the second primer partially anneals with the stage 2vector at Tm<40° C. and partially with Tu2 at Tm<40° C. In someembodiments, the total Tm for each primer is designed to be about 55-65°C., about 58-62° C. or about 60° C.

Also provided herein is a non-naturally occurring library of geneticmodules, comprising:

-   -   a plurality of pre-designed promoters,    -   a plurality of pre-designed untranslated regions,    -   a plurality of pre-designed terminators,    -   a plurality of pre-designed stage 1 vectors, and    -   at least one pre-designed stage 2 vector,    -   wherein each promoter, untranslated region, terminator, stage 1        vector and stage 2 vector are engineered to have a pair of        restriction sites of a first type IIs enzyme.

In some embodiments, the stage 1 and stage 2 vectors can each furthercomprise a pair of restriction sites of a second type IIs enzyme. Thefirst and/or second type IIs enzyme can be selected from BsaI, Eco31I,BspTN1, Bso31I, BbsI, BpuAI, BpiI, BstV21, BsmBI, Esp3I, FokI, AlwI, andBfilI. The first and second type IIs enzymes are different in certainembodiments.

A further aspect relates to a kit for in vitro assembly of geneticmodules, comprising:

-   -   a) any library of genetic modules disclosed herein, and    -   b) instruction for in vitro assembly of a coding sequence of        interest with a promoter, an untranslated region and a        terminator selected from the library into a transcription unit        in a stage 1 vector, and further assembly of a plurality of        transcription units into a stage 2 vector.

The method, library and kit disclosed herein can be used for rapidassembly of any genetic circuit of interest, or one or more portionsthereof. The assembled genetic circuit or portion thereof is compatiblewith the in vivo environment and thus, can be seamlessly transitionedfrom in vitro to in vivo testing.

BRIEF DESCRIPTION OF THE FIGURES

The presently disclosed technology will be further explained withreference to the attached drawings. The drawings shown are notnecessarily to scale, with emphasis instead generally being placed uponillustrating the principles of the presently disclosed technology.

FIG. 1 illustrates, in panel (a), a hypothetical circuit composed of ncomponents to be prototyped in vivo or in TX-TL, and in panel (b),conventional prototyping in vivo that requires the reduction of ncomponents to 3 plasmids, which can then be transformed into a cell.

FIG. 2A illustrates exemplary stage 0 pieces/genetic modules and theirpre-designed cohesive ends that dictate the assembly order of PUCT.

FIG. 2B illustrates exemplary cloning ends of stage 0 pieces forassembly into stage 1 vectors v1-1 and v1-2.

FIG. 3A illustrates an exemplary workflow of assembling modularcomponents into a plasmid.

FIG. 3B illustrates the different workflow, from an assembled plasmidfrom FIG. 3A, between traditional cloning and rapid assembly in vitro.

FIG. 3C illustrates an exemplary PCR strategy using specially designedprimers to selectively amplify correctly assembled plasmid from FIG. 3Ato prepare linear DNA for TX-TL.

FIG. 4A illustrates an exemplary workflow from stage 0 pieces to stage 1constructs, and the cycling between stage 1 and stage 2 constructs.

FIG. 4B illustrates, in one embodiment, the assembly of stage 0 pieceswith stage 1 vector into stage 1 construct.

FIG. 4C illustrates exemplary swappable areas on stage 1 vector toensure in vivo compatibility.

FIG. 4D illustrates an exemplary sticky end for a stage 1 vector.

FIG. 4E illustrates an exemplary cycling between stage 2 and stage 1indefinitely with set vectors.

FIG. 5A illustrates an exemplary assembly strategy of 3 transcriptionunits in 3 separate plasmids (each having different antibioticresistance markers and/or origins of replication), with possibility ofcycling to combine all 3 to 1 stage 2 plasmid and further cycling (notshown).

FIG. 5B illustrates an exemplary assembly strategy of 6 transcriptionunits (from 6 stage 1 constructs) into 1 stage 2 plasmid. Furthercycling is possible but not shown.

FIG. 5C illustrates an exemplary assembly strategy of 5 transcriptionunits into 1 vector (top) and 3 to 1 (bottom) with new vectors that usedifferent cohesive ends.

FIGS. 6A-6C illustrate, in one embodiment, assembly and cloning of twovariants of a 6-piece nested feed-forward loop.

FIG. 7A illustrates the testing of an exemplary circuit assembled usingthe present technology, a feed-forward loop and nested forward-loop, invitro in a cell-free transcription-translation system to determinecircuit performance.

FIG. 7B illustrates the screening of an exemplary plasmid containing onetranscription unit (in a stage 1 construct), by colony PCR, to determineplasmids for sequencing (not shown).

FIG. 7C illustrates, in one embodiment, the screening of 6 variants of afinal plasmid assembled using the present technology, containing 6transcription units (from 6 stage 1 constructs, into a stage 2construct) by plasmid size on a gel, with one plasmid (c1) out of 6correctly sized.

FIGS. 8A-8C illustrate, in one embodiment, assembly and testing of avariant of a 5-node oscillator. Specifically, FIG. 8A illustrates anassembly map of 5 stage 1 constructs into 1 stage 2 construct. FIG. 8Billustrates the testing of the stage 1 linear DNA constructs in vitro ina cell-free transcription-translation system. FIG. 8C illustrates thetesting of the stage 2 construct in vivo.

While the above-identified drawings set forth presently disclosedembodiments, other embodiments are also contemplated, as noted in thediscussion. This disclosure presents illustrative embodiments by way ofrepresentation and not limitation. Numerous other modifications andembodiments can be devised by those skilled in the art which fall withinthe scope and spirit of the principles of the presently disclosedembodiments.

DETAILED DESCRIPTION

Compositions and methods disclosed herein relate to methods, librariesand kits for rapid in vitro assembly of any genetic circuit of interest,or one or more portions (subcircuits) thereof. The assembled geneticcircuit or portion thereof is, by design, compatible with the in vivoenvironment. The technology disclosed herein therefore permits theseamlessly transition from in vitro to in vivo testing. Significantly,the rapid, entirely in vitro assembly technique disclosed herein can beused to assemble regulatory elements and basic circuits from standard orcustom pieces in, e.g., under 4 h, with complete testing in, e.g., under8 h. By maintaining an engineering cycle time of 8 h or less, thepresent technology enables prototyping of multicomponent circuits in astandard business day or less.

In contrast, conventional technology requires step-by-step cloning andtesting of each part of a multicomponent circuit, before the completecircuit can be cloned into a plasmid for propagation in vivo. This is alabor-intensive and serial process that has a 1-week testing cycle,which scales poorly for complex circuits (FIG. 1, panel a). Althoughlarge-scale successes have been accomplished by this testing method,there is a significant time cost to this engineering cycle. For example,the industrial production of artemisinin from synthetic circuits in E.coli and S. cerevisiae has taken 150 “person-years,” of which much timecan be attributed to part testing. As further illustrated in FIG. 1,panels (a) and (b), to initially test an n-part circuit in vivo wouldrequire log₃(n) rounds of plasmid cloning, assuming assemblies of 5pieces at the same time (four regulatory units plus a vector backbone).This restriction results from the carrying capacity of the cell of amaximum of 3 different plasmids to maintain a limited number ofantibiotic cassettes and origins of replication.

Using the rapid in vitro assembly approach disclosed herein, the presentdisclosure circumvents the conventional molecular cloning process thatis costly and labor intensive. Engineering-driven approaches and systemsare provided herein that significantly accelerate the design-build-testcycles required for reprogramming existing biological systems,constructing new biological systems and testing genetic circuits usefulin many areas including biology, engineering, green chemistry,agriculture and medicine.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., at least one) of the grammatical object of the article.By way of example, “an element” means one element or more than oneelement.

As used herein, the term “about” means within 20%, more preferablywithin 10% and most preferably within 5%. The term “substantially” meansmore than 50%, preferably more than 80%, and most preferably more than90% or 95%.

As used herein, “a plurality of” means more than 1, e.g., 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, e.g., 25,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or more, or anyinteger therebetween.

As used herein, the terms “nucleic acid,” “nucleic acid molecule” and“polynucleotide” may be used interchangeably and include bothsingle-stranded (ss) and double-stranded (ds) RNA, DNA and RNA:DNAhybrids. These terms are intended to include, but are not limited to, apolymeric form of nucleotides that may have various lengths, includingdeoxyribonucleotides and/or ribonucleotides, or analogs or modificationsthereof. A nucleic acid molecule may encode a full-length polypeptide orRNA or a fragment of any length thereof, or may be non-coding.

Nucleic acids can be naturally-occurring or synthetic polymeric forms ofnucleotides. The nucleic acid molecules of the present disclosure may beformed from naturally-occurring nucleotides, for example formingdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules.Alternatively, the naturally-occurring oligonucleotides may includestructural modifications to alter their properties, such as in peptidenucleic acids (PNA) or in locked nucleic acids (LNA). The terms shouldbe understood to include equivalents, analogs of either RNA or DNA madefrom nucleotide analogs and as applicable to the embodiment beingdescribed, single-stranded or double-stranded polynucleotides.Nucleotides useful in the disclosure include, for example,naturally-occurring nucleotides (for example, ribonucleotides ordeoxyribonucleotides), or natural or synthetic modifications ofnucleotides, or artificial bases. Modifications can also includephosphorothioated bases for increased stability.

“Assembly” or “assemble” means a process in which nucleic acid fragments(e.g., genetic modules as defined hereunder) are operably linked withone another in a pre-designed order to form a longer nucleic acidsequence. For example, genetic modules (also referred to as “stage 0pieces” or “pieces” in the context of assembly in some embodiments) canbe assembled into transcription units or parts. The transcription unitscan be present in linear format or in a circular plasmid, which issometimes referred to as “stage 1 constructs.” Two or more transcriptionunits can be assembled into a complete or partial circuit, sometimesreferred to as “stage 2 constructs.” In some embodiments, assembly canbe achieved using pre-selected cohesive ends that define thepre-designed order of genetic modules or transcription units in theassembled product. A first nucleic acid sequence is “operably linked”with a second nucleic acid sequence when the sequences are so arrangedthat the first nucleic acid sequence affects the function of the secondnucleic acid sequence. Preferably, the two sequences are part of asingle contiguous nucleic acid molecule and more preferably areadjacent. For example, a promoter is operably linked to a gene or acoding sequence if the promoter regulates or mediates transcription ofthe gene in a cell.

A “circuit” or “genetic circuit” as used herein refers to a collectionof parts (also referred to as “transcription units” or “Tu” in someembodiments) that undergo transcription and/or translation to producemRNA or proteins, respectively (each an “output” of the part). The partoutput can interact with other parts (for example to regulatetranscription or translation) or can interact with other molecules inthe cell (e.g., small molecules, DNA, RNA or proteins that are presentin the cellular environment). For example, a circuit can be a metabolicpathway or a genetic cascade, which can be naturally occurring ornon-naturally occurring, artificially engineered. Each part in thecircuit can include a set of components or genetic modules, e.g., apromoter, ribosome binding site (RBS), coding sequence (CDS) and/orterminator. These components may be interconnected or assembled indifferent ways to implement different parts, and the resultant parts maybe combined in different ways to create different circuits or pathways.In addition to these parts, the circuit may contain additional molecularspecies that are present in a cell or in the cell's environment that thecomponents interact with.

As described herein, “genetic module” and “genetic element” may be usedinterchangeably and refer to any coding and/or non-coding nucleic acidsequence. Genetic modules may be operons, genes, gene fragments,promoters, exons, introns, regulatory sequences, or any combinationthereof. In some embodiments, a genetic module refers to one or more ofcoding sequence, promoter, terminator, untranslated region, ribosomebinding site, polyadenlylation tail, leader, signal sequence, vector andany combination of the foregoing. In certain embodiments, a geneticmodule can be a transcription unit as defined herein.

Genetic modules may be derived from the genome of natural organisms orfrom synthetic polynucleotides or a combination thereof. In someembodiments, the genetic modules are derived from different organisms.Genetic modules useful for the methods described herein may be obtainedfrom a variety of sources such as, for example, DNA libraries, BAC(bacterial artificial chromosome) libraries, de novo chemical synthesis,commercial gene synthesis or excision and modification of a genomicsegment. The sequences obtained from such sources may then be modifiedusing standard molecular biology and/or recombinant DNA technology.Exemplary methods for modification of polynucleotide sequences include,for example, site directed mutagenesis; PCR mutagenesis; inserting,deleting or swapping portions of a sequence using restriction enzymesoptionally in combination with ligation; in vitro or in vivo homologousrecombination; and site-specific recombination; or various combinationsthereof. In other embodiments, the genetic sequences useful inaccordance with the methods described herein may be syntheticoligonucleotides or polynucleotides produced by any methods known in theart.

In some embodiments, genetic modules share less than 99%, less than 95%,less than 90%, less than 80%, or less than 70% sequence identity with anative or natural nucleic acid sequences. Identity can each bedetermined by comparing a position in each sequence which may be alignedfor purposes of comparison. When an equivalent position in the comparedsequences is occupied by the same base or amino acid, then the moleculesare identical at that position; when the equivalent site occupied by thesame or a similar amino acid residue (e.g., similar in steric and/orelectronic nature), then the molecules can be referred to as homologous(similar) at that position. Expression as a percentage of homology,similarity, or identity refers to a function of the number of identicalor similar amino acids at positions shared by the compared sequences.Expression as a percentage of homology, similarity, or identity refersto a function of the number of identical or similar amino acids atpositions shared by the compared sequences. Various alignment algorithmsand/or programs may be used, including FASTA, BLAST, or ENTREZ FASTA andBLAST are available as a part of the GCG sequence analysis package(University of Wisconsin, Madison, Wis.), and can be used with, e.g.,default settings. ENTREZ is available through the National Center forBiotechnology Information, National Library of Medicine, NationalInstitutes of Health, Bethesda, Md. In one embodiment, the percentidentity of two sequences can be determined by the GCG program with agap weight of 1, e.g., each amino acid gap is weighted as if it were asingle amino acid or nucleotide mismatch between the two sequences.Other techniques for alignment are described by Doolittle, MethodsEnzymol. 1996; 266:368-82. Preferably, an alignment program that permitsgaps in the sequence is utilized to align the sequences. TheSmith-Waterman is one type of algorithm that permits gaps in sequencealignments. Also, the GAP program using the Needleman and Wunschalignment method can be utilized to align sequences. An alternativesearch strategy uses MPSRCH software, which runs on a MASPAR computer.MPSRCH uses a Smith-Waterman algorithm to score sequences on a massivelyparallel computer.

A “library” of genetic modules refers to a collection of pre-made,standard genetic modules. The library can be pre-designed such that eachmodule therein has been engineered to generate compatible cohesive endsupon, e.g., restriction enzyme digestion. In one example, all geneticmodules within a library can be designed to be flanked by the samerestriction sites. Such an engineered library is non-naturallyoccurring.

As used herein, the term “coding sequence” or “CDS” refers to a nucleicacid that contains genetic information encoding a polypeptide, protein,or untranslated RNA (e.g., rRNA, tRNA, anti-sense RNA). Additionalelements such as promoter, terminator, 5′ untranslated region (UTR), and3′ UTR may be needed for the transcription and/or translation of thecoding sequence.

As used herein, the term “promoter” refers to a DNA sequence which whenligated to a nucleotide sequence of interest is capable of controllingthe transcription of the nucleotide sequence of interest into mRNA. Apromoter is typically, though not necessarily, located 5′ (i.e.,upstream) of a nucleotide sequence of interest whose transcription intomRNA it controls, and provides a site for specific binding by RNApolymerase and other transcription factors for initiation oftranscription. A promoter may be constitutively active (“constitutivepromoter”) or be controlled by other factors such as a chemical, heat orlight. The activity of an “inducible promoter” is induced by thepresence or absence or biotic or abiotic factors. Commonly usedconstitutive promoters include CMV, EF1a, SV40, PGK1, Ubc, human betaactin, CAG, Ac5, Polyhedrin, TEF1, GDS, ADH1 (repressed by ethanol),CaMV35S, Ubi, H1, U6, T7 (requires T7 RNA polymerase), and SP6 (requiresSP6 RNA polymerase). Common inducible promoters include TRE (inducibleby Tetracycline or its derivatives; repressible by TetR repressor), GAL1& GAL10 (inducible with galactose; repressible with glucose), lac(constitutive in the absence of lac repressor (LacI); can be induced byIPTG or lactose), T7lac (hybrid of T7 and lac; requires T7 RNApolymerase which is also controlled by lac operator; can be induced byIPTG or lactose), araBAD (inducible by arabinose which binds repressorAraC to switch it to activate transcription; repressed cataboliterepression in the presence of glucose via the CAP binding site or bycompetitive binding of the anti-inducer fucose), trp (repressible bytryptophan upon binding with TrpR repressor), tac (hybrid of lac andtrp; regulated like the lac promoter; e.g., tacI and tacII), and pL(temperature regulated). The promoter can be a prokaryotic or eukaryoticpromoter, depending on the host. Common promoters and their sequencesare well known in the art.

One should appreciate that promoters have modular architecture and thatthe modular architecture may be altered. Bacterial promoters typicallyinclude a core promoter element and additional promoter elements. Thecore promoter refers to the minimal portion of the promoter required toinitiate transcription. A core promoter includes a Transcription StartSite, a binding site for RNA polymerases and general transcriptionfactor binding sites. The “transcription start site” refers to the firstnucleotide to be transcribed and is designated +1. Nucleotidesdownstream of the start site are numbered +1, +2, etc., and nucleotidesupstream of the start site are numbered −1, −2, etc. Additional promoterelements are located 5′ (i.e., typically 30-250 bp upstream of the startsite) of the core promoter and regulate the frequency of thetranscription. The proximal promoter elements and the distal promoterelements constitute specific transcription factor site. In prokaryotes,a core promoter usually includes two consensus sequences, a −10 sequenceor a −35 sequence, which are recognized by sigma factors. The −10sequence (10 bp upstream from the first transcribed nucleotide) istypically about 6 nucleotides in length and is typically made up of thenucleotides adenosine and thymidine (also known as the Pribnow box). Thepresence of this box is essential to the start of the transcription. The−35 sequence of a core promoter is typically about 6 nucleotides inlength. The nucleotide sequence of the −35 sequence is typically made upof the each of the four nucleosides. The presence of this sequenceallows a very high transcription rate. In some embodiments, the −10 andthe −35 sequences are spaced by about 17 nucleotides. Eukaryoticpromoters are more diverse than prokaryotic promoters and may be locatedseveral kilobases upstream of the transcription starting site. Someeukaryotic promoters contain a TATA box, which is located typicallywithin 40 to 120 bases of the transcriptional start site. One or moreupstream activation sequences (UAS), which are recognized by specificbinding proteins can act as activators of the transcription. Theses UASsequences are typically found upstream of the transcription initiationsite. The distance between the UAS sequences and the TATA box is highlyvariable and may be up to 1 kb.

“Untranslated region” or “UTR” refers to either section of theuntranslated portion in an mRNA molecule that is located at the 5′ side(“5′ UTR”) or 3′ side (“3′ UTR”) of a coding sequence. The 5′ UTRcontains a sequence that is recognized by the ribosome which allows theribosome to bind and initiate translation (“ribosome binding site” or“RBS”). The 3′ UTR is involved in translation termination as well aspost transcriptional gene expression.

“Terminator” refers to a nucleic acid sequence that hinders or stopstranscription of a RNA polymerase. Generally a self-annealing hairpinstructure may be formed on the elongating transcript, which results inthe disruption of the mRNA-DNA-RNA polymerase ternary complex. Thenatural terminator sequence contains a 20 base pair GC-rich region ofdyad symmetry followed by a short poly-T tract which is transcribed toRNA to form the terminating hairpin and a 7-9 nucleotide “U track”respectively. (Dyad symmetry refers generally to two areas of a DNAstrand whose base pair sequences are inverted repeats of each other.They are often described as palindromes.) A survey of natural andsynthetic terminators is provided in Chen et al., Characterization of582 natural and synthetic terminators and quantification of their designconstraints, Nature Methods 10, 659-664 (2013), incorporated herein byreference.

As used herein, the term “vector” refers to any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, artificial chromosome,episome, virus, virion, etc., capable of replication when associatedwith the proper control elements and which can transfer gene sequencesinto or between cells. The vector may contain a selection modulesuitable for use in the identification of transformed or transfectedcells. For example, selection modules may provide antibiotic resistant,fluorescent, enzymatic, as well as other traits. As a second example,selection modules may complement auxotrophic deficiencies or supplycritical nutrients not in the culture media. Types of vectors includecloning and expression vectors. As used herein, the term “cloningvector” refers to a plasmid or phage DNA or other DNA sequence which isable to replicate autonomously in a host cell and which is characterizedby one or a small number of restriction endonuclease recognition sitesand/or sites for site-specific recombination. A foreign DNA fragment maybe spliced into the vector at these sites in order to bring about thereplication and cloning of the fragment. The term “expression vector”refers to a vector which is capable of expressing of a gene that hasbeen cloned into it. Such expression can occur after transformation intoa host cell, or in an in vitro system. The cloned DNA is usuallyoperably linked to one or more regulatory sequences, such as promoters,activator/repressor binding sites, terminators, enhancers and the like.The promoter sequences can be constitutive, inducible and/orrepressible.

A vector used in assembly of stage 1 constructs is referred to as a“stage 1 vector” in some embodiments. A vector used in assembly of stage2 constructs is referred to as a “stage 2 vector” in some embodiments.

As used herein, unless otherwise stated, the term “transcription” refersto the synthesis of RNA from a DNA template; the term “translation”refers to the synthesis of a polypeptide from an mRNA template.Translation in general is regulated by the sequence and structure of the5′ untranslated region (5′-UTR) of the mRNA transcript. One regulatorysequence is the ribosome binding site (RBS), which promotes efficientand accurate translation of mRNA. The prokaryotic RBS is theShine-Dalgarno sequence, a purine-rich sequence of 5′-UTR that iscomplementary to the UCCU core sequence of the 3′-end of 16S rRNA(located within the 30S small ribosomal subunit). Various Shine-Dalgarnosequences have been found in prokaryotic mRNAs and generally lie about10 nucleotides upstream from the AUG start codon. Activity of a RBS canbe influenced by the length and nucleotide composition of the spacerseparating the RBS and the initiator AUG. In eukaryotes, the Kozaksequence lies within a short 5′ untranslated region and directstranslation of mRNA. An mRNA lacking the Kozak consensus sequence mayalso be translated efficiently in an in vitro system if it possesses amoderately long 5′-UTR that lacks stable secondary structure. While E.coli ribosome preferentially recognizes the Shine-Dalgarno sequence,eukaryotic ribosomes (such as those found in retic lysate) canefficiently use either the Shine-Dalgarno or the Kozak ribosomal bindingsites.

“Type IIs enzyme” refers to restriction endonucleases that recognize adouble-stranded DNA at a specific sequence (“restriction site” or“recognition site”) and cleave the double-stranded DNA at a cleavagesite that is outside the recognition site on the double-stranded DNA.Generally overhangs of from 3 to 6 nucleotides are produced upon typeIIs restriction. A selection of such enzymes is provided on the REBASEwebpage (rebase.neb.com/cqi-bin/asvmmlist) and in the review ofSzybalsky et at, 1991, Gene, 100:13-26. Examples include but are notlimited to BstF5I, BtsCI, BsrDI, BtsI, AlwI, BccI, BsmAI, EarI, PleI,BmrI, BsaI, BsmBI, FauI, Mn1I, SapI, BbsI, BciVI, HphI, MboII, BfuAI,BspCNI, BspMI, SfaNI, HgaI, BseRI, BbvI, EciI, FokI, BceAI, BsmFI,BtgZI, BpuEI, BsgI, MmeI, BseGI, Bse3DI, BseMI, AcIWI, Alw261, Bst6I,BstMAI, Eam1104I, Ksp6321, PpsI, BfiI, Bso31I, BspTNI, Eco31I, Esp3I,SmuI, BfuI, BpiI, BpuAI, BstV2I, AsuHPI, Acc361, LweI, AarI, BseMII,TspDTI, TspGWI, BseXI, BstV1I, Eco571, Eco57MI, GsuI, and BcgI. Thoselisted on pages 12-13, Table 1 and Table 2 of U.S. Publication No.20130267021 are non-exclusive examples and are incorporated herein byreference.

As used herein, the term “host” or “host cell” refers to any prokaryoticor eukaryotic single cell (e.g., yeast, bacterial, archaeal, etc.) cellor organism. The host cell can be a recipient of a replicable expressionvector, cloning vector or any heterologous nucleic acid molecule. Hostcells may be prokaryotic cells such as species of the genus Escherichiaor Lactobacillus, or eukaryotic single cell organism such as yeast. Theheterologous nucleic acid molecule may contain, but is not limited to, asequence of interest, a transcriptional regulatory sequence (such as apromoter, enhancer, repressor, and the like) and/or an origin ofreplication. As used herein, the terms “host,” “host cell,” “recombinanthost” and “recombinant host cell” may be used interchangeably. Forexamples of such hosts, see Green & Sambrook, 2012, Molecular Cloning: Alaboratory manual, 4th ed., Cold Spring Harbor Laboratory Press, NewYork, incorporated herein by reference.

One or more nucleic acid sequences can be targeted for delivery totarget prokaryotic or eukaryotic cells via conventional transformationtechniques. As used herein, the term “transformation” is intended torefer to a variety of art-recognized techniques for introducing anexogenous nucleic acid sequence (e.g., DNA) into a target cell,including calcium phosphate or calcium chloride co-precipitation,conjugation, electroporation, sonoporation, optoporation, injection andthe like. Suitable transformation media include, but are not limited to,water, CaCl₂, cationic polymers, lipids, and the like. Suitablematerials and methods for transforming target cells can be found inGreen & Sambrook, 2012, Molecular Cloning: A laboratory manual, 4th ed,Cold Spring Harbor Laboratory Press, New York, incorporated herein byreference, and other laboratory manuals.

As used herein, the term “selectable marker” or “reporter” refers to agene, operon, or protein that upon expression in a host cell ororganism, can confer certain characteristics that can be relativelyeasily selected, identified and/or measured. Reporter genes are oftenused as an indication of whether a certain gene has been introduced intoor expressed in the host cell or organism. Examples of commonly usedreporters include: antibiotic resistance (“abR”) genes, fluorescentproteins, auxotropic selection modules, β-galactosidase (encoded by thebacterial gene lacZ), luciferase (from lightning bugs), chloramphenicolacetyltransferase (CAT; from bacteria), GUS (β-glucuronidase; commonlyused in plants) green fluorescent protein (GFP; from jelly fish), andred fluorescent protein (RFP). Typically host cells expressing theselectable marker are protected from a selective agent that is toxic orinhibitory to cell growth.

The term “engineer,” “engineering” or “engineered,” as used herein,refers to genetic manipulation or modification of biomolecules such asDNA, RNA and/or protein, or like technique commonly known in thebiotechnology art.

Other terms used in the fields of recombinant nucleic acid technologyand molecular and cell biology as used herein will be generallyunderstood by one of ordinary skill in the applicable arts.

Assembly Method and Kit

Methods and kits for rapidly assembling pre-made DNA modules aredescribed herein to run rapid (e.g., 8 hours or less) prototyping invitro and systemically enable in vivo testing using the existing DNApieces. In some embodiments, the method is also referred to as“Iterative Assembly” or “Idiotproof Assembly.” The method can usemodular components, sometimes referred to as Iterative Assembly_Promoter(IA_P), IA_Untranslated Region (IA_UTR), IA_Coding Sequence (IA_CDS),IA_terminator (IA_T), and IA_vector (IA_V). There are also variants ofthese modules, such as IA_PUCT (a combination of all modules) and IA_UC(a combination of UTR and CDS). Variations of the modules can be usedfor DNA modules that do not fit the mold of a traditional IA_P, IA_U,IA_C, or IA_T. For example, a random spacer may be implemented asIA_PUCT while a RNA-based activator as IA_UC. An example of these piecescan be found under FIG. 2A, which are also referred to as “stage 0pieces” in some embodiments. It should be noted that exemplary cohesiveends are shown in FIG. 2A for each piece to illustrate the design inwhich the identity of the cohesive ends dictate the order of assemble,IA_P followed by IA_U, then IA_C and IA_T. Other cohesive ends ofdifferent length and/or sequence can also be used. In the case of a4-nucleotide cohesive end, 4^4=256 different cohesive ends areavailable.

IA_P, IA_U, IA_C, IA_T, or a combination thereof may be assembled in apredetermined order by having predefined cloning ends attached orengineered to 5′ and/or 3′ ends. One exemplary set of cloning ends areillustrated in FIG. 2B and listed below (only top strand sequences areshown below since bottom strands are complementary to top strands).

SEQ ID No. Cloning End Top Strand Sequence 1 v1-1, 3′ endGAAGACAACCACGCATAGAGACCAGGAC 2 V1-2, 3′ end GAAGACAACATAGCATAGAGACCCACCT3 Promoter, 5′ end AGAACGGTCTCAGCAT 4 Promoter, 3′ end AAGCTGAGACCTTACG5 UTR1, 5′ end AGCCAGGTCTCAAAGC 6 UTR1, 3′ end AATGTGAGACCGGGGA 7CDS, 5′ end AACAGGGTCTCAAATG 8 CDS, 3′ end TGAATGAGACCACTAA 9Terminator, 5′ end GGCTCGGTCTCATGAA 10 Terminator, 3′ endGTCGTGAGACCCGGAC 11 v1-1, 5′ end ATATAGGTCTCTGTCGGGCATTGTCTTC 12v1-2, 5′ end TAGCGGGTCTCTGTCGTGCCTTGTCTTC

The above sequences are for illustration purpose only. It should benoted that the sequence of the cloning ends for any genetic module canbe varied, e.g., by replacing the BsaI and/or BbsI recognition siteswith other Type IIs enzyme sites, and/or replacing the spacer sequencelocated between the Type IIs enzyme recognition site and cleavage sitewith any sequence (e.g., degenerate sequence). Suitable Type IIs enzymesinclude but are not limited to BsaI, Eco31I, BspTN1, Bso31I, BbsI,BpuAI, BpiI, BstV21, BsmBI, Esp3I, FokI, AlwI, and BfilI.

While the identity of the cloning ends can vary, the cohesive endsgenerated by Type IIs enzyme digestion must be designed in a way suchthat each module can only fit in a certain position in the assembledproduct. In other words, it is important that only an IA_P can anneal toan IA_U, an IA_C to an IA_T (and not an IA_P to an IA_T), or any otherorder that ensures that the genetic modules are operably linked to oneanother to allow, e.g., transcription and/or translation. However, theends can use any ligation method provided the previous statement holdstrue. In the example shown in FIG. 2B, the Type IIs BsaI enzyme is used(recognition sites are indicated by solid lines and cleavage sites areindicated by arrows) and Golden Gate Assembly is conducted; the IA_P toIA_U linker is AAGC, IA_U to IA_C is AATG, IA_C to IA_T is TGAA, IA_V toIA_P is GCAT, and IA_T to IA_V is GTCG. Any other Type IIs enzyme can beused in lieu of BasI.

In some embodiments, compatible cohesive ends that anneal with eachother can be assembled together using Golden Gate Assembly, as disclosedby Engler et al., PLoS One, 4(5), e5553, incorporated herein byreference in its entirety.

Per module, there can be many IA_P, IA_U, IA_C, IA_T and IA_V stage 0pieces. These can be collected in a library for future use. In someembodiments, the library can be stored in multi-well plates and can beused as part of a kit for rapid in vitro assembly of desirabletranscription units or genetic circuits. As of April 2016, there are 96IA_P modules, 48 IA_U modules, 99 IA_C modules, 22 IA_T modules in acustom library made. Each one of these modules can be combined to oneanother, thereby giving 96*48*99*22=10 million combinations, each atranscription unit. Stage 0 pieces can be engineered to have predefinedcloning ends (e.g., those in FIG. 2B), which can either be added on bypolymerase chain reaction or can be put in by cloning into a predefinedvector.

Each IA_P, IA_U, IA_C, and IA_T can be assembled with a vector, IA_V. Asshown in FIG. 2B, 2 different vectors (v1-1 and v1-2) having differentorigin of replication and/or selectable marker can be used. As of April2016, a total of 49 vectors have been made in a custom library. Thevectors are chosen to facilitate end in vivo testing (stage 1 vectorsand stage 2 vectors discussed in more detail hereunder). The speed ofrunning in vitro partially comes from the ability to reuse modules andfrom mixing and matching modules.

FIGS. 3A-3B demonstrate an exemplary general workflow. Referring to FIG.3A, the assembly procedure proceeds by, e.g., standard Golden GateAssembly (GGA) to a pre-defined vector, which creates minimal amounts ofplasmid DNA copies. The ligation is a 1-pot ligation, e.g., all pieces(not pre-digested) can be added in and digestion and ligation will occurwithin the reaction. This plasmid DNA can then be transformed to preparefor in vivo testing as well as used after a PCR reaction to conduct invitro testing, as shown in FIG. 3B.

For the in vitro testing, after the assembly reaction linear DNA can bemade (FIG. 3C). Two primers P1 and P2 can be designed such that eachpartially binds to the vector at one portion and partially to thepromoter/terminator at another portion (overlap primers), each portionwith a melting temperature (Tm)<40° C., but both portions togetherprovide a total Tm of about 55-65° C., about 58-62° C. or about 60° C.It is designed this way to enforce enrichment of correctly clonedconstructs, and provides a correctly-sized linear DNA which can be thenbe run in TX-TL. If a construct is not correctly cloned (e.g., IA_V doesnot ligate to IA_P and/or IA_T does not ligate to IA_V), one primer willbind with a low Tm and will fail the PCR. Once a linear DNA is enriched,it can then be run in TX-TL with the support of gamS protein to blockexonuclease activity.

Although one can use non-overlap primers to amplify the linear DNA totest by designing primers to bind to vectors only, this alternativeapproach will also amplify the case where the vector self-ligates(without ligating with the genetic modules) or the genetic modules donot anneal in the desired correct order. Therefore, the use ofnon-overlap primers may result in poor selection of the correct linearDNA cassette. Using overlap primers as described herein is especiallycritical when multiple ligation reactions are required where theligation efficiency may be low.

Linear DNA produced by PCR may not be completely of correct sequenceidentity due to mutations introduced during the PCR amplification stepsor during the digestion and ligation step. However, this can bemitigated by the fact that the linear DNA can be run in an in vitrotranscription-translation system in non-clonal form, as there is norequirement to provide clonal DNA in the in vitro expression reaction.

If running a plasmid is desired in lieu of running a linear DNA, one canfollow the “Traditional Cloning” workflow in FIG. 3B to purify plasmidwhich can be sequence or size verified and run in TX-TL.

A challenge for transitioning large circuits from in vitro to in vivo isthe difficulty in consolidating all pieces onto vivo compatibleplasmids. Using the method described here, this consolidation isextremely easy and requires little to no DNA design. Once stage 0 piecesare determined they can be cloned into different IA_V to produce stage 1constructs that can be already ready for vivo expression or can becycled to make stage 2 constructs (FIG. 4A). Stage 1 vectors can be thesame vectors used to produce the rapid linear DNA from FIG. 3C. Anexample of the production of the stage 1 construct from stage 0 piecesand a stage 1 vector is given in FIG. 4B.

FIG. 4C shows that the stage 1 vectors can have swappable areas toensure in vivo compatibility. In particular, the origin of replications(ORIs) can be swapped and the antibiotic resistance markers (abR) can beswapped to those commonly used. Any origins of replication commonly usedin molecular cloning may be used, such as colE1, pSC101, p15A, pBBR1,pMB1 and R6K. The antibiotic resistance marker can be any markercommonly used in molecular cloning, such as AmpR/CarbR(ampicillin/carbenicillin resistance), KanR (kanamycin resistance), CmR(chloramphenicol resistance), ZeoR (zeocin resistance), TetR(tetracycline resistance), SpecR (spectinomycin resistance), StrepR(streptomycin resistance), NeoR (neomycin resistance), and BleR(bleomycin resistance). Different vectors having different ORI and/orabR can be pre-made and collected in a custom library.

Additionally, there are sticky ends (solid boxes) engineered in thevectors that, in the example shown in FIG. 4C, are cut with a differentenzyme than the stage 0 to stage 1 transition (e.g., BbsI instead ofBsaI). An exemplary DNA sequence (top strand: GAAGACAACCACGCAT (SEQ IDNo.: 13) and sticky end for BbsI digestion and ligation in vector v1-1are shown in FIG. 4D.

FIG. 4E then demonstrates the cycling ability between stage 1 pieces andstage 2 pieces. While FIG. 4E illustrates a 2 piece to 1 piece assembly,one can also go from multiple pieces (e.g., 3, 4, 5, 6, 7, 8, 9 or more)to 1 piece. In this example, 2 PUCT transcription units (stage 0 pieces)are put into two different stage 1 vectors with BsaI digestion, v1-1 andv1-2, to make stage 1 constructs. These stage 1 constructs havecompatible ends with each other, and can be directly put into a one-potassembly with a stage 2 vector (v2-1 or v2-2 in this case) to form stage2 constructs when cut with BbsI. Note here each stage 2 construct has 2PUCT transcription units (not shown). In this case, both stage 2constructs can regenerate stage 1 constructs by another one-pot assemblywith a recycled stage 1 vector (v1-1 or v1-2) using BsaI digestion.Then, by going through 3 cycles of assembly (1 cycle of stage 0 to stage1, 1 cycle of stage 1 to stage 2, and 1 cycle of stage 2 to stage 1) onecan make up to 8 PUCT transcription units. FIGS. 6A-6C show thisstrategy in one specific example, aiming to combine 2×6 PUCT expressibleunits into 2 plasmids for in vivo expression, as explained in moredetail below.

FIG. 4E also demonstrates that the orientation of the PUCT transcriptionunits can be varied depending on the needs of the final product and thestability of the DNA. In this example, v1-2 is designed to flip theorientation of the second PUCT unit relative to the first PUCT unit(e.g., divergent orientation). However, the PUCT unit can be engineeredto be convergent or divergent. In some embodiments, multiple invertedrepeats or repeated DNA segments may cause hairpins upon plasmidpropagation in vivo and subsequent deletions, which may requireconvergent or divergent assembly. In certain embodiments, it may also bedesirable to choose different assembly directions to influencetranscriptional strength of the resulting PUCT unit due to secondarystructure or context effects.

Note that as long as DNA originates in plasmid form, it has been foundthat sequencing of the constructs is not required. The product ifverified to be of the correct size from one stage can go directly intothe latter stage. If the DNA originates in linear form or is formed fromsynthetic DNA, sequencing can be optionally used to rule out mutationsintroduced by the DNA polymerase amplification step.

FIG. 5A shows another exemplary layout, where 3 PUCT transcription unitsare cloned into 3 separate stage 1 plasmids to make stage 1 constructswhich are in vivo compatible (e.g., colE1/ampR, p15A/cmR, pSC101*/kanR).These plasmids can then be tested immediately in TX-TL or in vivo. Ifcombining them onto 1 plasmid is desired, the plasmids can be assembledin a one-pot reaction with, e.g., BbsI to make a stage 2 construct whichhas all 3 PUCT transcription units in 1 plasmid. This again can betested in TX-TL or in vivo.

FIG. 5B shows another exemplary layout, where 6 PUCT transcriptionunits, from 6 separate stage 1 vectors, are cloned into a stage 2construct (using stage 2 vectors, e.g., v2-1, v2-2, v2-3, v2-4, v2-4a).ORI and abR are shown for each stage 2 vector. Generally it isimpossible to express 6 separate plasmids in vivo since the maximumcompatibility of host cells typically only allows for 3 plasmids. Here,it is shown in FIG. 5B that each stage 1 vector can be different (withdifferent ORI and/or abR as shown), as long as the sticky ends areconserved. Again, the sticky ends are for illustration purpose only anddifferent sequences can be used. The destination stage 2 vector can alsobe varied depending on the need (e.g., low copy/medium copy plasmid ordifferent antibiotic resistance marker).

It is noted in the exemplary layout of FIG. 5B that by designatingdifferent options for each stage 1 vector, one can expediteconsolidating multiple PUCT transcription units into one unit withoutthe need to conduct significant re-engineering and cloning. For example,if it is desired to produce a circuit using 6 PUCT units to be tested incombinations in vivo and then combined into one unit, vectors can bechosen such that the stage 1 constructs are individually compatible fortesting (e.g., having compatible OR1 and/or abR, such as colE1 AmpR andP15A CmR, to co-transform and test). In addition, the stage 2 vector canbe chosen from a family of vectors (e.g., from a premade library),depending on the need for the final testing environment (e.g., low-copyor high-copy number, and/or certain origin of replication to becompatible with an additional plasmid to be co-transformed).

In some cases, the post-cloned PUCT in a stage 1 or stage 2 vector maybe toxic to the cell at high copy number or high expression level, andproduce a subsequent deletion of a regulatory region (e.g., promoter,UTR) after propagation in vivo. Thus, in certain embodiments, adifferent vector may be selected with different copy numbers to reducedeletion phenotypes.

The exemplary layout of FIG. 5B demonstrates that new stage 1 and stage2 vectors can also be engineered depending on the needs of the user(e.g., high copy or low copy number, or different antibiotic resistancemarker).

It is noted that the efficiency of digestion and ligation of PUCTtranscription units may change as a function of number of PUCT unitsligated together, length of PUCT units, and secondary structure of PUCTunits. Therefore, in some embodiments, the assembly strategy may bedesigned to compensate for, e.g., a decrease in efficiency of ligationby, e.g., increasing digestion and ligation cycles, selecting forsmaller colonies after transformation, and/or utilizing lower-copy finalvectors to reduce expression load.

If 6 transcription units are not available at the same time, one caneasily scale down by changing the end sticky end on the stage 2 vector.Shown in FIG. 5C is an example using 5 PUCT transcription units and anexample using 3 PUCT expressible units. One can also replace PUCTexpressible units with randomly generated DNA to act as filler.

To demonstrate the power of the assembly method disclosed herein, one ofordinary skill in the art will appreciate that by using, e.g., FIG. 5B'sassembly method, if 18 PUCT transcription units were made, within twostages this assembly method would allow for 3 in vivo compatibleplasmids using only two cycles of cloning. This could take only 1 weekor less (as opposed to the current standard of multiple months inconventional cloning), and would be sufficient to express the largestknown synthetic circuits in E. coli (Moon, Lou, Tamsir, Stanton, &Voigt, 2012). In addition, the assembly needs only minimal planningbefore implementation and does not require re-engineering of vectorsafter cloning. By dramatically reducing the number of cloning cycles,this procedure saves significant time and requires less user know-how inorder to assemble complete circuits. It also allows for the testing ofintermediate plasmids in an alternative system, such as an in vitrocell-free transcription-translation system.

It should also be noted that the present technology is different thanthe GoldenBraid assembly disclosed by Sarrion-Perdigones et al. (2011)PLoS ONE 6(7): e21622, doi:10.1371/journal.pone.0021622, incorporatedherein by reference. GoldenBraid by design only permits binary assembly,i.e., joining of two modules, using an automated process. Thus, for 8transcription units it takes 3 cycles to get into 1 plasmid usingGoldenBraid. In contrast, the present technology significantly limitsthe number of cycles needed (e.g., only 1 or 2 cycles needed) byallowing more than 2 modules to be assembled into one module. While lessof an “automated” process compared to GoldenBraid and requiring moreadvanced planning, the present technology achieves significant time andlabor savings. For example, the efficient ligation of 6 large PUCT units(each of ˜1-2 kb) and a vector (˜2-3 kb) into one unit (˜8-15 kb) isdemonstrated in the present technology.

The method outlined herein is additionally different from GoldenBraid byproviding flexibility in choosing intermediate vectors to be compatiblefor end-test conditions, such that modules of the circuit can be testedindependently or in combination in vitro or in vivo without interruptingthe complete circuit assembly process. The in vitro testing issignificant, as the GoldenBraid technology is optimized entirely for invivo expression of the final assembly (which takes significant time tocomplete). Therefore, the present technology allows for testing andre-engineering of the circuit during the engineering process, instead ofrequiring the entire circuit be completed before implementation.

The present technology can be used in connection with the “design,build, test” (DBT) cycle for prototyping and debugging a biomolecularcircuit as disclosed in U.S. patent application Ser. No. 15/046,374filed Feb. 17, 2016, entitled “CELL-FREE BIOMOLECULAR BREADBOARDS ANDRELATED METHODS AND ARRANGEMENTS”, the disclosure of which isincorporated herein by reference. For example, following rational designand model of a circuit or pathway comprising a plurality of parts, theindividual parts can be built and combined to form the designed circuitor subcircuits for in vitro testing as follows:

-   -   A specific concentration of each part is mixed in a container        containing extract and buffer, such as the TX-TL system        disclosed herein. Different concentrations of different parts        can be used to create a collection of variants that will be        tested.    -   Additional chemicals can be added to the container to establish        different conditions under which the part is to be        characterized. For example, inducers may be added in different        concentrations to characterize the function of a repressor        protein.    -   The container is heated to a temperature an incubated for a        period of time.    -   A measurement is taken, using an optical assay (absorbance or        fluorescence), a chemical assay (mass spec) or other analytical        technique.    -   The previous two steps are iterated at a specific rate and for a        duration of time.

Optionally, to build confidence in the in vitro results, an in silicoapproach can also be used that uses as an input characterized parts andsimulates, in silico, different formulations of the parts to form thedesign circuit or subcircuits. This is done most accurately to reflectthe findings one would obtain in vitro; however, the in silico toolboxcan also provide data on the predicted function of the circuit in vivo.

Using the measurement data from in vitro testing, the performance of thecircuit can be characterized by analyzing the data and determining whichcombinations of parts, and in what relative concentrations, whencombined together implement the function that was designed originally.At the end of this step, a single DBT cycle is complete. At this stage,if the circuit does not perform as designed, the data from this step andprevious steps can be used to redesign the circuit, returning to anyprior step in the workflow.

Once a specific combination of parts has been determined to provide thedesigned function in the breadboard, the parts can be combined so thatmultiple parts are assembled on pieces of DNA compatible with the cellusing the present technology. Often this DNA is a circularized moleculereferred to as a plasmid. Typically, the circuit is consolidated onto 1,2 or 3 plasmids that have compatible origins of replications (e.g., cansurvive in the cell together). In this form, the circuit can be testedboth in vitro (using the cell-free system) and in vivo (using a cell).

The following steps can be used to create the plasmid form of thecircuit that will be verified:

-   -   The specific parts can be designed to be in the stoichiometric        ratios determined to be the most effective in vitro to match in        vivo. E.g., items that require low expression are put on low        copy number plasmids whereas those that require high expression        which are put on high copy number plasmids. Promoter or ribosome        binding site (RBS) can also be varied in strengths to modulate        expression level.    -   The individual parts are then engineered to form stage 1        constructs, using Golden Gate Assembly or any other suitable        assembly methods.    -   All parts for a designed circuit can then be engineered to form        stage 2 constructs (e.g., on 1-3 plasmids) such that the final        plasmids can be directly transformed into a cell for        verification, without the need for further cloning.

In vitro verification can include the following steps:

-   -   A specific concentration of the plasmid or other DNA sequence        that contains multiple parts is mixed in a container containing        extract and buffer such as the TX-TL system disclosed herein. If        more than one piece of DNA is used, different concentrations of        different plasmids can be used to create a collection of        variants that will be tested.    -   Additional chemicals can be added to the container to establish        different conditions under which the part is to be        characterized. For example, inducers may be added in different        concentrations to characterize the function of a repressor        protein.    -   The container is heated to a temperature an incubated for a        period of time.    -   A measurement is taken, using an optical assay (absorbance or        fluorescence), a chemical assay (mass spec) or other analytical        technique.    -   The previous two steps are iterated at a specific rate and for a        duration of time.

The output from the in vitro verification step can be a set of data thatmeasure the performance of the circuit under desired conditions in acell-free environment. These data are compared to the desired operationof the circuit (as represented by the initial design and model step). Ifthe results are the same, the circuit is operational in an in vitroenvironment. Depending on the application, this in vitro version of thecircuit can be used directly in applications. If the output from thisstep does not match the model, the data from the in vitro verificationstep and previous steps can be used to redesign the circuit, returningto any prior step in the workflow.

In vivo verification can include the following steps:

-   -   Cells are chemically, electrically or thermally treated to allow        them to transport DNA from their external environment into the        cytoplasm of the cell.    -   Plasmids containing the DNA implementing a circuit, instantiated        on one or more (e.g., 1-3) plasmids, are introduced into the        environment of the treated cells.    -   The plasmids are transformed into the cells by the introduction        of an environmental stimulus (e.g., temperature) that causes at        least a fraction of the cells to incorporate one or more        plasmids into the cytoplasm.    -   The cells are transferred to container that contains growth        media and a selecting agent (e.g., an antibiotic), such that        only those cells containing the desired circuit elements can        divide and grow.    -   The container is heated to a temperature and incubated for a        period of time.    -   A measurement is taken, using an optical assay (absorbance or        fluorescence), a chemical assay (mass spec) or other analytical        technique.    -   The previous two steps are iterated at a specific rate and for a        duration of time.

The output from the in vivo verification step can be a set of data thatmeasure the performance of the circuit under desired conditions in acell. These data are compared to the desired operation of the circuit(as represented by the design and model step). If the results are thesame, the circuit is operational in an in vivo environment.

EXAMPLES Example 1: Assembly of a Nested Feed-Forward Loop

FIGS. 6A-6C illustrates the cloning of two variants of a 6-piece nestedfeed-forward loop. In FIG. 6A, a promoter, UTR (and/or) coding sequence,and terminator are first incorporated into a “PUCT” transcriptionconstruct over 6 different plasmids (stage 1 constructs). For examplefor construct 362p, a “P” unit, “UC” unit, and “T” unit are cloned intovector 10-2 in a one-pot reaction by digestion with BsaI. Then, stillreferring to FIG. 6A, the 6 constructs on 6 different plasmids areconsolidated onto two different plasmids (stage 2 constructs 411 in v2-3and 412 in v2-4), where the digestion of v10-2, v10-3b, and v1-1 withBbsI allow cloning into v2-3 or v-2-4 in a one-pot reaction. Forexample, constructs 362p, 363p, 287p and v2-3 are combined together toassemble plasmid 411 in v2-3. Stage 2 constructs 411 in v2-3 and 412 inv2-4 can be directly tested in vivo with, e.g., IPTG induction in JW0336cells.

FIG. 6B shows the same procedure for different variants of the nestedfeed-forward loop, from 6 different stage 1 constructs to 2 stage 2constructs, 413p in v2-3 and 414p in v2-4.

In the final stage shown in FIG. 6C, 411p in v2-3 and 412p in v2-4 arecombined together with vector v1-4 and BsaI in a one-pot reaction toproduce 415p v1-4, which contains the 6-transcription unit circuit. 413pin v2-3 and 414p in v2-4 are combined to produce 416p v1-4. Finalconstructs 415p v1-4 and 416p v1-4 can be tested directly in vivo withIPTG/aTc/3OC6/3OC12 in JW0336 cells.

It is worth noting that the vectors are designed beforehand tofacilitate compatibility. In addition, all constructs in the samevectors are interchangeable. For example, 362p, 364p, 334p, or 367p areall in v10-2 and can be used interchangeably.

In specific, in FIG. 6A 362p contains a small trigger RNA from Green etal. (Cell 2014 Nov. 6; 159(4):925-39) that is controlled by a stronglambda-phage (POr21Pr) constitutive promoter, repressible in thepresence of a lambda-phage repressor (lambda-CI). 363p has aconstitutive promoter, J23150, in front of a lacI repressor, that isnormally “off” but can be activated by expression of the trigger1 RNAfrom 362p. 287p has a lac promoter (that can be repressed by lacIrepressor) that encodes for a sfGFP (reporter) tagged with an ssrAdegradation tag, that is normally “off” but can be activated byexpression of the trigger1 RNA from 362p. This module is independent,i.e., the three plasmids are functional by themselves. This moduleproduces a pulse in vivo, e.g., an increase in signal by reporting fromthe sfGFP in 287p, followed by a decrease in signal as lad is producedfrom 363p to shut off expression of 287p. sfGFP is then degraded byClpXP AAA+ proteases or and/or diluted from cellular growth anddivision.

Because the module is independent, 362p, 363p, and 287p are purposelychosen to be on compatible vectors (cmR p15A, kanR pSC101, ampR colE1)such that they can be co-transformed in vivo for testing, while stillable to be directly used in the second assembly reaction.

A separate module, on the bottom of FIG. 6A, is composed of 334p, 368pand 369p. 334p has a lac promoter (that can be repressed by ladrepressor) that encodes for a lasR that is normally “off” but can beactivated by expression of the trigger1 RNA from 362p. 368p has a laspromoter (that can be activated by lasR from 334p) in front of a tetRcoding sequence. 369p has a las promoter with a built in tetO1 operator,that can be repressed by tetR produced by 368p, and expresses mRFP withan ssrA degradation tag. This module, when activated by the top module,also produces a pulse in vivo of mRFP. It is nested, as the secondfeed-forward loop (334p, 368p, 369p) requires activation from the firstfeed-forward loop (362p, 363p, 287p).

362p in FIG. 6A is assembled by the following protocol: v10-2 (2217 bp241 ngul 66 ng): 0.28 uL, P52 Or2-Or1-Pr1-short, short attachment (90 bp53 ngul 2 ng): 0.51 uL at 1:10 dil, UC10 green_trigger1_1stgen_17 bp(AGCA) (98 bp 222 ngul 2 ng): 1.33 uL at 1:100 dil, T17 T500_noGap,short attachment, (AGCA) (70 bp 204 ngul 2 ng): 1.03 uL at 1:100 dil,BSA at 10x: 1.00 uL, T4 ligase Buffer: 1.00 uL, BsaI/HF: 0.67 uL, T4Ligase 2 mil units: 0.67 uL, ddH20: 3.52 uL. Assembly conditions were: 3min at 37° C., 4 min at 16° C., cycled 25 times; followed by 5 min at50° C., 5 min at 80° C.

1 uL of the assembly reaction for 362p is then transformed into a JM109competent sub-cloning strain following a published protocol from ZymoMix-and-Go JM109 chemical transformation. After transformation, cellsare recovered for 1 hour at 37° C. in SOC media, and then plated onchloramphenicol-resistant LB plates for overnight growth. This strain ischosen, because although expression is driven by a strong POr21Prpromoter, the trigger RNA itself is non-toxic. If the expressible unitis toxic, then an alternate strain can be chosen (e.g., a KL740 strainfrom the Yale E. coli genetic stock center) to repress expression.

In lieu, 362p and other DNAs can also be amplified by PCR and testedimmediately in vitro in linear form, or can be transformed, miniprepped,and tested in vitro or in vivo in plasmid form. FIG. 7A showsrepresentative data that would be collected by testing rapid assemblylinear DNA pieces from this module in vitro. In FIG. 7A, top, is ademonstration that a specific trigger (trigger 1) can activate aspecific switch (switch 1) in vitro in a dose-dependent manner. In FIG.7A, bottom, is a demonstration that linear DNA can be used to implementthe whole circuit in vitro. The circuit responds to increased IPTG byincreasing signal production as lad repression is lifted.

Resulting individual colonies undergo colony PCR using primers bindingto the v10-2 vector (TTCTCATGTTTGACAGCTTATCA (SEQ ID NO.: 14),ATAACTCAAAAAATACGCCCG (SEQ ID NO.: 15)) that are expected to produce a354 bp construct. As demonstrated in FIG. 7B, c1 and c3-c6 producecorrect sized bands against a 2 log ladder (NEB N3200), while c2 is anincorrect band and likely ligation of an empty vector. The successfulproduction of 362p is confirmed by Sanger Sequencing (not shown).

Each of 363p, 287p, 364p, 365p, 367p, 368p, 369p is prepared similarlyas 362p using the same procedure, but varying the individual P, U, (UC),C, or T pieces and resistance of selection.

362p, 363p, and 287p can then be assembled into 411p in FIG. 6A by thefollowing protocol: v2-3: 2568 bp/203 ngul at 35.1 ng→0.17 uL; 362p:32.3 ng, 2363 bp, 40 ngul→0.81 uL; 363p: 66 ng, 4833 bp, 33 ngul→2 uL;287p: 45.8 ng, 3352 bp, 482 ngul→1 uL of 1:10 dil, BSA at 10x-1 uL, T4Ligase−1 uL, BbsI−0.66 uL, T4 Ligase 2 mil−0.66 uL, water−2.7 uL.Assembly conditions were: 3 min at 37° C., 4 min at 16° C., cycled 25times; followed by 5 min at 50° C., 5 min at 80° C. 411p is thentransformed into a JM109 strain, plated on spectinomycin-resistant LBplates, grown, screened by colony PCR, and sequenced as done for 362p.

We note that 411p assembles 362p, 363p, and 287p in a stage 2 vectorv2-3 that is specR p15A. This is chosen purposely, as specR does notshare the resistance marker of 362p, 363p, or 287p (to avoid backgroundselection from transformed original plasmids during the selection). Inaddition, cmR and p15A allow this plasmid to be tested in vivo withanother plasmid, 361p, which produces additional trigger RNA and isampR, colE1.

412p is prepared similarly as 411p but using 334p, 368p, 369p, and v2-4.

415p in FIG. 6C is the final testing plasmid and can be assembled by thefollowing protocol: 411p: 5020 bp, 68 ngul, 62 ng→0.91 uL, 412p: 5312bp, 79 ngul, 66 ng→0.84 uL, v1-4, 2217 bp, 267 ngul, 28 ng→1.05 uL 1:10dil, Bsa-10x→1 uL, T4L Buffer→1 uL, BsaI-HF→0.66 uL, T4L→0.66 uL,H20→3.88 uL. Assembly conditions were: 3 min at 37° C., 4 min at 16° C.,cycled 25 times; followed by 5 min at 50° C., 5 min at 80° C. 415p isthen transformed into a JM109 strain, plated onchloramphenicol-resistant LB plates, grown, screened, and sequenced.

Note that for larger plasmids such as 415p (7,461 bp) and 416p (8,308p),a significant metabolic load may be introduced into the cell that willslow growth. In addition, the efficiency of ligation may be reduced.Therefore, it may be necessary to choose smaller or slower-growingcolonies, and to screen additional constructs. In this example, for 416pcolonies were grown and miniprepped to determine size rather than bycolony PCR. The resulting plasmids (416p clones c1 to c6) are shown inFIG. 7C, compared to a supercoiled DNA ladder (NEB N0472) and 2 logladder (NEB N3200). In this example, only 416p-c1 is of the correctsize.

415p and 416p are considered the final circuit (with 415p combining 6PUCT transcriptional units and 416p combining 6 PUCT transcriptionalunits) and can be used for testing in vivo in a final testing strain,such as JW0336 (Yale E. coli genetic stock center).

It should be noted that while in this example, the first feed-forwardloop (362p, 363p, 287p) and the second feed-forward loop (334p, 368p,369p) are assembled in a 2-step process into one plasmid, one skilled inthe art would appreciate that all 6 transcription units can be assembledtogether in a 1-step assembly using the present technology.

Example 2: Assembly of an Oscillator Plasmid

FIG. 8A illustrates the assembly of a 5 PUCT transcriptional unitoscillator plasmid into a final stage 2 vector, v41-2. In FIG. 8A (top),5 individual PUCT transcriptional units (490p, 567p, 492p, 568p, and569p) are assembled into compatible stage 1 constructs (v40-1, v50-2,v40-3a, v50-4, v40-5, respectively). These 5 plasmids can then beassembled with a v41-2 vector in a one-pot reaction with BbsI Type IIsrestriction enzyme to generate the final 5n2 plasmid.

490p is a SrpR-ssrA repressor protein driven by a PhlF promoter.SrpR-ssrA represses the promoter on 567p, which is a BetI-ssrA repressordriven by a SrpR promoter. BetI-ssrA represses the promoter on 568p,which is a QacR-ssrA repressor driven by a BetI promoter. QacR-ssrArepresses the promoter on 569p, which is a TetR-ssrA repressor driven bya QacR promoter. TetR prepresses the promoter on 492p, which is aPhlF-ssrA repressor driven by a TetR promoter. PhlF represses 490p. Thisring of repression produces in an in vitro transcription-translationsystem an oscillating waveform over time, when the output is read on anyaxis (e.g. pTetR-Cerulean, pQacR-Citrine) (FIG. 8B). Note that this invitro expression is done with 5 strands of linear DNA and 2 reporterplasmids; the linear DNA can be produced immediately from the assemblyprocedure described in this technology. The 5 plasmids (490p, 567p,492p, 568p, and 569p), when combined into one plasmid (5n2) andtransformed into a JS006 strain (Nature. 2008 Nov. 27; 456(7221):516-9)with a reporter plasmid such as pPhlF-BCD22-sfGFP-ssrA(LAA) (ampR,colE1), produce a corresponding waveform in vivo over time (FIG. 8C).

As an exemplary stage 1 plasmid, 567p, P65U18C64T14 v50-2, is assembledby combining: v50-2 (3647 bp 47 ngul 66 ng): 1.42 uL, P65 pSrpR,Stanton_14 (115 bp 16 ngul 2 ng): 1.31 uL at 1:10 dil, U18 BCD7(AATG)(141 bp 68 ngul 2 ng): 0.38 uL at 1:10 dil, C64 betI-ssrA (AATG), orig.E. coli (650 bp 40 ngul 11 ng): 2.97 uL at 1:10 dil, T14 ECK120033736(164.6x), short attachment (95 bp 187 ngul 1 ng): 0.93 uL at 1:100 dil,BSA at 10x: 1.00 uL, T4ligase Buffer: 1.00 uL, BsaI/HF: 0.67 uL,T4Ligase 2 mil units: 0.67 uL. Assembly conditions were: 3 min at 37°C., 4 min at 16° C., cycled 25 times; followed by 5 min at 50° C., 5 minat 80° C.

1 uL of the assembly reaction for 567p is then transformed into a JM109competent sub-cloning strain following a published protocol from ZymoMix-and-Go JM109 chemical transformation. After transformation, cellswere plated on carbenicillin-resistant LB plates for overnight growth.Colonies can then be isolated for sequencing and verification.

Note that 490p, 567p, 568p, and 569p are purposely chosen to betransformed into a pSC101 AmpR backbone, as each of these promoters(pPhlF, pSrpR, pBetI, and pQacR, respectively) do not have easilyaccessible strains that have repressors to repress expression.Therefore, a low-copy pSC101 vector is preferable to avoid toxicity.However, 492p is under control of a pTetR promoter, which can berepressed by tetR overexpressing strains such as MG1655Z1. Therefore, ifusing the MG1655Z1 strain, a high-copy colE1 vector can be used.

In lieu, each post-assembled, pre-transformed plasmid and other DNAs canalso be amplified by PCR and tested immediately in vitro in linear form,or can be transformed, miniprepped, and tested in vitro or in vivo inplasmid form. FIG. 8B is a demonstration of a circuit run off ofcomparably produced linear DNA.

To make the stage 2 plasmid 5n2, assembled are: V41-2: 3581 bp, 64 ngul,52 ng→0.81 uL, 567: 30 ngul, 4483 bp, 65 ng→2.17 uL, 568: 34 ngul, 4450bp, 65 ng→1.91 uL, 569: 66 ngul, 4555 bp, 66 ng→1 uL, 490 old: 4555 bp,172 ngul, 66 ng→0.38 uL, 492: 3204 bp, 501 ngul, 46 ng→0.28 uL 1:3 dil,Bsa10x: 1 uL, T4L: 1 uL, BbsI: 0.66 uL, T4Ligase: 0.66 uL, H2o: 0.13 uL.Assembly conditions were: 3 min at 37° C., 4 min at 16° C., cycled 25times; followed by 5 min at 50° C., 5 min at 80° C. 5n2 is thentransformed into a JM109 strain, plated on kanamycin-resistant LBplates, grown, screened, and sequenced.

5n2 is a very large plasmid (8014 bp) using 5 active PUCTtranscriptional units; therefore a significant metabolic load may beintroduced into the cell that will slow growth. In addition, theefficiency of ligation may be reduced. Therefore, it may be desirable tochoose smaller or slower-growing colonies, and to screen additionalconstructs.

Materials and Methods

Cell-free expression preparation and execution: Preparation of thecell-free TX-TL expression system was done according to previouslydescribed protocols, resulting in extract with conditions: 8.9-9.9 mg/mLprotein, 4.5-10.5 mM Mg-glutamate, 40-160 mM K-glutamate, 0.33-3.33 mMDTT, 1.5 mM each amino acid except leucine, 1.25 mM leucine, 50 mMHEPES, 1.5 mM ATP and GTP, 0.9 mM CTP and UTP, 0.2 mg/mL tRNA, 0.26 mMCoA, 0.33 mM NAD, 0.75 mM cAMP, 0.068 mM folinic acid, 1 mM spermidine,30 mM 3-PGA, 2% PEG-8000.14 Unless otherwise specified, one extract set“e10” was used consistently throughout the experiments to preventvariation from batch to batch and to test feed-forward loop circuits invitro. Extract “eZS4” was similarly prepared for oscillator in vitrowork. Extract “eZS4” was prepared using above conditions but using aJS006 starting strain. TX-TL reactions were conducted in a volume of 10μL in a 384-well plate (Nunc) at 29° C., using a three tube system:extract, buffer, and DNA. When possible, inducers such as IPTG orpurified proteins such as gamS were added to a mix of extract and bufferto ensure uniform distribution. When using a plate reader, for deGFP,samples were read in a Synergy H1 plate reader (Biotek) using settingsfor excitation/emission: 485 nm/525 nm, gain 61. For mRFP, settings were580 nm/610 nm, gain 61 or 100. All samples were read in the same platereader, and for deGFP and mRFP rfu units were converted to μM of proteinusing a purified deGFP-His6 standard and purified mRFP standard. Unlessotherwise stated, end point measurements are after 8 h of expression at29° C.

Cell-free in vitro execution of feed-forward loop: Cell-free experimentstesting individual switches were run with 8 nM of a rapid assemblylinear DNA product of pLac-switch (from Green et al.2014)-sfGFP-ssrA-ECK120029600 and 4 nM-32 nM of rapid assembly linearDNA products pTet-trigger-T500, where three separate triggers are testedand one is known to activate the switch tested in vivo. Reactions arealso run with gamS at 3.5 uM, and IPTG of 1 mM. Cell-free experimentstesting whole circuits were run with 32 nM of a rapid assembly linearDNA product of pTet-trigger-T500, 8 nM of a rapid assembly linear DNAproduct of pLac-switch-sfGFP-ssrA-ECK120029600, and 4 nM of a rapidassembly linear DNA product of pOR21Pr-switch-lacI-ECK120033736.Reactions are also run with gamS at 3.5 uM, and varying concentrationsof IPTG.

GamS Protein Purification: The composition of buffers used was asfollows: buffer L, 50 mM Tris-Cl pH 8, 500 mM NaCl, 5 mM imidazole, 0.1%Triton X; buffer W, 50 mM Tris-Cl pH 8, 500 mM NaCl, 25 mM imidazole;buffer E, 50 mM Tris-Cl pH 8, 500 mM NaCl, 250 mM imidazole; buffer S,50 mM Tris-Cl pH 7.5, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 2% DMSO. Afrozen stock of P_araBAD-gamS in a BL21-DE3 E. coli strain was grownovernight in LB-carbenicillin media. 100 mL was used to inoculate 1 LLB-carbenicillin to an OD 600 nm of 0.4-0.6 at 37° C., 220 rpm. Cellswere then incubated to 0.25% arabinose (final concentration) and grownfor four additional hours at 25 C, 220 rpm, before being pelleted andfrozen at −80° C. Cells were resuspended in buffer L, mechanically lysedand incubated with Ni-NTA agarose (Qiagen). Ni-NTA agarose was washedtwice with 15 column volumes of buffer W and eluted in buffer E.Fractions with a ˜13 kD band were concentrated and dialyzed into bufferS overnight and further purified on a 26/60 Sephadex 75 column. Proteinconcentration was verified by Bradford, concentrated to 3 mg/mL using anUltra-0.5 3K MWCO Centrifugal Filter (Ambion), and stored in buffer S at−80° C. Protein purity was verified by gel. Purification steps wereverified by SDS-PAGE gel electrophoresis.

Plasmid DNA and PCR Product Preparation: Plasmids used in this studywere constructed using standard cloning procedures and maintained in aKL740 strain if using an OR2-OR1 promoter (29° C.), a MG1655Z1 strain ifusing a Pl-tetO1 or Pl-lacO1 promoter, a BL21-DE3 strain for proteinpurification, a BL21 strain for promoter characterization, or a JM109strain for all other constructs. KL740 upregulates a temperaturesensitive lambda cI repressor, and MG1655Z1 upregulates tetR and lad.PCR products were amplified using Pfu Phusion Polymerase (New EnglandBiolabs) for all constructs, and were DpnI digested. Plasmids wereeither miniprepped using a PureYield column (Promega) or midipreppedusing a NucleoBond Xtra Midi column (Macherey-Nagel). All plasmids wereprocessed at stationery phase. Before use in the cell-free reaction,both plasmids and PCR products underwent an additional PCR purificationstep using a QiaQuick column (Qiagen), which removed excess saltdetrimental to TX-TL, and were eluted and stored in 10 mM Tris-Clsolution, pH 8.5 at 4° C. for short-term storage and −20° C. forlong-term storage.

In vitro Linear DNA Assembly. Linear DNA fragments were amplified usingPfu Phusion Polymerase (New England Biolabs), DpnI digested for 5 min at37° C. (New England Biolabs) while verified with agarose gelelectrophoresis, and PCR purified using previously described procedures.Fragments were then assembled in vitro using either isothermal assemblyor Golden Gate assembly. For isothermal assembly, Gibson Assembly MasterMix (New England Biolabs) was used according to manufacturerinstructions with 1:3 molar ratio vector/insert, and reacted at 1 h at50° C. For Golden Gate assembly, a 15 μL reaction was set up consistingof equimolar amounts of vector and insert, 1.5 μL 10×NEB T4 Buffer (NewEngland Biolabs), 1.5 μL 10×BSA (New England Biolabs), 1 μL BsaI (NewEngland Biolabs), and 1 μL T4 Ligase at 2 million units/mL (New EnglandBiolabs). Reactions were run in a thermocycler at either 10 cycles of 2min/37° C., 3 min/20° C., 1 cycle 5 min/50° C., 5 min/80° C. or 25cycles of 3 min/37° C., 4 min/16° C., 1 cycle 5 min/50° C., 5 min/80° C.For Golden Gate assembly, constructs with internal BsaI or BbsI cutsites were silently mutated beforehand using a QuikChange LightningMulti Site-Directed Mutagenesis kit (Agilent).

Rapid Assembly Product Protocol. The in vitro linear DNA assemblyprotocol was followed. Overlap primers were then designed to bind overthe vector:promoter and vector/terminator junctions such that the Tm ofbinding on each junction side was below 40° C. Then, 1 μL of theresulting assembly product was PCR amplified for 35 cycles in a 50 μLPCR reaction, and verified by agarose gel electrophoresis. If theresulting band was 80% or more pure, the DNA was PCR purified usingpreviously described procedures and used directly in TX-TL.

Protein purification: For fluorescent proteins eGFP, mRFP, and Venus andvariants eGFP-ssrA, mRFP-ssrA, and Venus-ssrA, coding sequences werecloned into a T7-lacO inducible vector containing a N-terminus His6 tagusing standard techniques and propagated in a BL21-DE3 strain (NewEngland Biolabs). Proteins were purified following a similar protocol asin Hodgman et al., Metab Eng, 2012. 14(3): p. 261-9, but were grown inTB broth in lieu of LB broth, induced with 1 mM IPTG (finalconcentration), and selected for a band between 25 kDa-35 kDacorresponding to the fluorescent protein in question. Fluorescentproteins were further processed in a Supradex 20 10/300 column to selectfor pure, active proportions, and flash-frozen at −80° C. in a storagebuffer consisting of: 50 mM Tris-Cl pH 7.5, 100 mM NaCl, 1 mM DTT, 1 mMEDTA, 2% DMSO. Final concentrations were: deGFP-ssrA, 164.8 uM; deGFP,184.8 uM; mRFP-ssrA, 185.6 uM; mRFP, 170.6 uM; Venus-ssrA, 87.9 uM;Venus, 147.5 uM.

In vivo strain preparation and testing for feed-forward loops. For invivo assays, plasmids were cloned into compatible vectors and chemicallytransformed into a compatible strain, such as JW0336. For single strainassays, cells were selected for on antibiotic resistant agar platesbefore use. For multi-panel assays, cells were recovered for 2 hours at29 C. in SOC medium (Sigma) before outgrowth at a 1.25% dilution inMOPS-EZ Rich (Teknova) 0.4% glycerol selective media containing 10 μg/mLchloramphenicol, 50 μg/mL kanamycin, 100 μg/mL carbenicillin, 100 μg/mLspectinomycin, or equivalent antibiotic dependent on strain and plasmidand storage at −80 C. To conduct the in vivo assays, cells were grown inthe same selective MOPS media to stationery phase at 29° C. Cells werethen diluted 1% into 500 uL per well in 96-well MatriPlates (Brooks LifeSciences) with half-antibiotic concentration previously used. Threeplate readers were used (Biotek H1/MF), which were calibrated forfluorescent intensity and absorbance. Plates were measured every 6minutes at deGFP, 485 nm/515 nm gain 61 and 100, and OD, 600 nm under alinear continuous shaking mode. At OD 0.1-0.2, cells were induced withappropriate small molecule (such as aTc, IPTG, or 3OC12HSL) and thenmeasured for an additional 16 hours until stationery phase.

Steady-state cell-free in vitro testing for oscillators: Experimentswere performed in a microfluidic nano-reactor device as described inNiederholtmeyer et al., PNAS 2013 vol. 110 no. 40 15985-15990, with somemodifications to optimize the conditions for the lysate-based TX-TL mix.Reaction temperature was 33° C. Lysate was diluted to 2× of the finalconcentration in 5 mM HEPES 5 mM NaCl buffer (pH 7.2). The reactionbuffer mix was combined with template DNA and brought to a finalconcentration of 2×. For a 24 h experiment 30 μl of these stocks wereprepared. During the experiment, lysate and buffer/DNA solutions werekept in separate tubing feeding onto the chip, cooled to approximately6° C., and combined on-chip. The experiments were run with dilutionrates (μ) between approximately 2.8 and 0.5 h⁻¹, which corresponds todilution times, t_(d)=ln(2)μ⁻¹, between 15 and 85 min. These wereachieved with dilution steps exchanging between 7 and 25% of the reactorvolume with time intervals of 7 to 10 min, which alternately added freshlysate stock or fresh buffer/DNA solution into the reactors. Dilutionrates were calibrated before each experiment. DNA templateconcentrations used in steady-state reactions for 5n2 are:pBetI-BCD7-QacR-ssrA(LAA), 1 nM Linear; pPhlF-BCD7-SrpR-ssrA(LAA), 12 nMLinear; pQacR-BCD7-TetR-ssrA(LAA), 4 nM Linear;pSrpR-BCD7-BetI-ssrA(LAA), 24 nM Linear; pTetR-BCD7-PhlF-ssrA(LAA), 4 nMLinear; pTetR-Cerulean(ASV), 2.5 nM Plasmid, pQacR-BCD7-Citrine, 2.5 nMPlasmid. Arbitrary fluorescence values were converted to absoluteconcentrations from a calibration using purified Citrine, Cerulean, andmCherry.

In vivo testing for oscillators: Mother machine experiments wereconducted with custom-made microfluidic chips. E. coli cells weretrapped in channels of 30 μm length, 2 μm width and 1.2 μm height.Before loading onto the device, cells were grown from a frozen stock tostationery phase. Cells were then concentrated 10-fold and loaded ontothe chip. Experiments were performed using LB medium supplemented with0.075% Tween-20 at a flow rate of 400 μl/h. Oscillation traces werecollected from single mother machine traps using the backgroundsubtracted average fluorescence intensity of the entire trap. The straintested was 5n2 co-transformed with pPhlF-BCD22-sfGFP-ssrA(LAA) intoJS006.

EQUIVALENTS

The present disclosure provides among other things methods and systemsfor rapid in vitro assembly of genetic modules. While specificembodiments of the subject disclosure have been discussed, the abovespecification is illustrative and not restrictive. Many variations ofthe disclosure will become apparent to those skilled in the art uponreview of this specification. The full scope of the disclosure should bedetermined by reference to the claims, along with their full scope ofequivalents, and the specification, along with such variations.

INCORPORATION BY REFERENCE

The ASCII text file submitted herewith via EFS-Web, entitled“165948_010200_sequence.txt” created on Apr. 26, 2016, having a size of2,812 bytes, is hereby incorporated by reference in its entirety.

All publications, patents and sequence database entries mentioned hereinare hereby incorporated by reference in their entirety as if eachindividual publication or patent was specifically and individuallyindicated to be incorporated by reference.

REFERENCES

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The invention claimed is:
 1. A method for in vitro assembly of geneticmodules, comprising: (a) providing recombinant transcription units Tu1,Tu2 and TuN wherein N>=3, each recombinant transcription unit beingpresent in a separate stage 1 vector and flanked by a first pair ofrestriction sites of a first type IIs enzyme, wherein the first pair ofrestriction sites for each recombinant transcription unit arepre-designed such that upon digestion by the first type IIs enzyme,compatible cohesive ends are generated to allow ligation of therecombinant transcription units into a linear DNA molecule having apredetermined order 5′-Tu1-TuN-Tu2-3′; (b) providing a stage 2 vectorhaving a second pair of restriction sites of the first type IIs enzyme,wherein the second pair of restriction sites are pre-designed such thatupon digestion by the first type IIs enzyme, a first and second cohesiveend are generated to allow direct ligation of the first cohesive endwith Tu1 at its 5′ end and direct ligation of the second cohesive endwith Tu2 at its 3′ end; and (c) assembling the recombinant transcriptionunits and the stage 2 vector into a plasmid in a one-pot reactioncomprising the first type IIs enzyme and a ligase.
 2. The method ofclaim 1, wherein N=<9.
 3. The method of claim 1, wherein the first typeIIs enzyme is selected from BsaI, Eco31I, BspTN1, Bso31I, BbsI, BpuAI,BpiI, BstV21, BsmBI, Esp3I, FokI, AlwI, and BfilI.
 4. The method ofclaim 1, further comprising constructing each recombinant transcriptionunit from a promoter, an untranslated region, a coding sequence and aterminator, wherein one or more of the promoter, untranslated region,coding sequence and terminator are provided from a library of modularcomponents.
 5. The method of claim 4, wherein each modular componentcomprises flanking restriction sites of a second type IIs enzyme.
 6. Themethod of claim 5, wherein the second type IIs enzyme is selected fromBsaI, Eco31I, BspTN1, Bso31I, BbsI, BpuAI, BpiI, BstV21, BsmBI, Esp3I,FokI, AlwI, and BfilI.
 7. The method of claim 4, further comprisingselecting the one or more of the promoter, untranslated region, codingsequence and terminator from the library.
 8. The method of claim 1,wherein each stage 1 vector comprises a different origin of replicationand/or a different selectable marker.
 9. The method of claim 8, whereinthe origin of replication is selected from colE1, pSC101, p15A, pBBR1,pMB1 and R6K.
 10. The method of claim 8, wherein the selectable markeris selected from AmpR, KanR, CmR, ZeoR, TetR, SpecR, StrepR, NeoR, andBleR.
 11. The method of claim 1, wherein the stage 2 vector furthercomprises a pair of restriction sites of a second type IIs enzyme thatflank the second pair of restriction sites of the first type IIs enzyme.12. The method of claim 1, further comprising cycling the recombinanttranscription units between the stage 1 and stage 2 vectors to produce 2or more copies of the recombinant transcription units.
 13. The method ofclaim 1, further comprising providing two or more stage 2 vectors instep (b), and assembling in step (c) the recombinant transcription unitsand the two or more stage 2 vectors into two or more plasmids.
 14. Themethod of claim 13, wherein each stage 2 vector comprises a differentorigin of replication and/or a different selectable marker.
 15. Themethod of claim 13, further comprising assembling in step (c) up tothree plasmids.
 16. The method of claim 1, further comprising subjectingthe plasmid to expression.
 17. The method of claim 16, furthercomprising subjecting the plasmid to expression in an in vitrotranscription-translation system.
 18. The method of claim 17, furthercomprising amplifying the recombinant transcription units in apolymerase chain reaction prior to expression.
 19. The method of claim18, further comprising providing a first and second primer that span thefirst and second cohesive end, respectively, wherein the first primerpartially anneals with the stage 2 vector at Tm<40° C. and partiallywith Tu1 at Tm<40° C., and the second primer partially anneals with thestage 2 vector at Tm<40° C. and partially with Tu2 at Tm<40° C.
 20. Themethod of claim 16, further comprising subjecting the plasmid to in vivoexpression following transformation into a host cell.